FRET Imaging of Molecular Hierarchy at Subcellular Levels in Mechanotransduction Atherosclerosis is the leading cause of death in the United States and most other developed countries. Atherosclerosis occurs preferentially at vascular curvature and branch sites where the vessel walls are exposed to disturbed flow, but not at the straight parts of vessels where laminar flow dominates. Disturbed flows, in comparison to laminar flows, can also cause slower wound healing process following the balloon injury after atherosclerosis. Evidence has shown that the shear stresses resulting from the flows play crucial roles in regulating vascular endothelial cells (ECs), and subsequently endothelium permeability and atherosclerosis, wound healing and restenosis. It remains unclear, however, on how ECs sense the spatiotemporal characteristics of these mechanical stimuli and coordinate the molecular hierarchy at sub-cellular levels to determine patho-physiological consequences. Fluorescence resonance energy transfer (FRET) technology and genetically encoded biosensors have provided powerful tools for visualizing active molecular events with high spatiotemporal resolutions in live cells. In this proposal, we will apply multi-color FRET biosensors for the visualization of molecular hierarchies participating in Src signaling pathways at sub-cellular levels under different flows. Src can be activated by shear stress and plays central roles in a variety of cellular processes. We hypothesized that different flows can induce a RhoA/actin-dependent activation of Src with distinct spatiotemporal patterns. The activated Src can enhance the MLCK-mediated actomyosin contractility to modulate adherens junctions (AJs), which can affect endothelium permeability and consequently atherosclerosis. The flow-activated Src can also control the p130cas-Rac pathway, leading to the modulated EC protrusion, motility, wound healing process, and potentially restenosis. To test our hypothesis, three specific aims are proposed: (1) To visualize the spatiotemporal activation patterns of Src at sub-cellular membrane compartments under different flow patterns and examine the roles of RhoA and actin cytoskeleton in mediating these Src activations; (2) To dissect the roles of Src and MLCK in mediating the modulation of EC contractility and AJs in response to different flows; (3) To investigate the role of Src in regulating the p130cas, Rac, and EC protrusion under different flows. FRET biosensors with distinct colors will be developed to allow the simultaneous visualization of multiple molecular activities at sub-cellular levels in a single live cell. Pharmacological inhibitors, siRNAs, and genetic mutants will be employed to assess the roles of different signaling molecules. The Integration of multi-color FRET biosensors for the simultaneous visualization of multiple molecular events will significantly advance our systematic understanding of the molecular mechanism by which different flows affect cardiovascular diseases, such as atherosclerosis and restenosis. The newly developed biosensors will also provide powerful tools for detecting cardiovascular diseases as well as the efficacy of therapeutic inhibitors. PUBLIC HEALTH RELEVANCE: Narrative: Endothelial cells (ECs) are continuously exposed to flow and its resultant shear stress. It has been known that shear stress plays crucial roles in regulating EC function and the ensuing patho-physiological processes, such as atherosclerosis and restenosis. It is not clear, however, on how the cells perceive shear stress and coordinate molecular functions. This proposal will integrate the cutting-edge FRET technology and novel fluorescence proteins to visualize multiple active molecular events with high spatiotemporal resolutions in live ECs. The results should shed new light and advance our systematic understanding of the molecular mechanism of mechanotransduction and the ensuing pathophysiological processes. Therefore, the success of the proposed project will have significant impact on improving public health.